Use of acid stable protease in animal feed

ABSTRACT

The present invention relates to acid-stable proteases homologous to those derived from strains of the genus  Nocardiopsis , their use in animal feed, feed-additives and feed compositions containing such proteases, and methods for the treatment of vegetable proteins using such proteases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional of U.S. application Ser. No. 10/713,394filed on Nov. 14, 2003 (now U.S. Pat. No. 7,658,965), which is acontinuation of application Ser. No. 09/779,323 filed Feb. 8, 2001 (nowU.S. Pat. No. 6,855,548), which claims priority or the benefit under 35U.S.C. 119 of Danish application no. PA 2000 00200 filed Feb. 8, 2000and U.S. provisional application no. 60/183,133 filed Feb. 17, 2000, thecontents of which are fully incorporated herein by reference.

SEQUENCE LISTING

The present application contains a computer-readable form of a sequencelisting, which is fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of acid-stable proteases inanimal feed (in vivo), and to the use of such proteases for treatingvegetable proteins (in vitro).

Proteins are essential nutritional factors for animals and humans. Mostlivestock and many human beings get the necessary proteins fromvegetable protein sources. Important vegetable protein sources are e.g.oilseed crops, legumes and cereals.

When e.g. soybean meal is included in the feed of mono-gastric animalssuch as pigs and poultry, a significant proportion of the soybean mealsolids is not digested. For example, the apparent ileal proteindigestibility in piglets and growing pigs is only around 80%.

The stomach of mono-gastric animals and many fish exhibits a stronglyacidic pH. Most of the protein digestion, however, occurs in the smallintestine. A need therefore exists for an acid-stable protease that cansurvive passage of the stomach.

2. State of the Art

The use of proteases in animal feed, or to treat vegetable proteins, isknown from the following documents:

WO 95/28850 discloses i.a. an animal feed additive comprising a phytaseand a proteolytic enzyme. Various proteolytic enzymes are specified atp. 7.

WO 96/05739 discloses an enzyme feed additive comprising xylanase and aprotease. Suitable proteases are listed at p. 25.

WO 95/02044 discloses i.a. proteases derived from Aspergillus aculeatus,as well as the use in animal feed thereof.

U.S. Pat. No. 3,966,971 discloses a process of obtaining protein from avegetable protein source by treatment with an acid phytase andoptionally a proteolytic enzyme. Suitable proteases are specified incolumn 2.

U.S. Pat. Nos. 4,073,884, 5,047,240, 3,868,448, and 3,823,072, and3,683,069 describe protease preparations derived from various strains ofStreptomyces and their use in animal feed.

These proteases, however, are not acid-stable and/or are not homologousto the to proteases described herein.

SUMMARY OF THE INVENTION

Proteases have now been identified which are found to be veryacid-stable, and expectedly of an improved performance in animal feed.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to theaccompanying drawings, in which:

FIG. 1 shows pH-stability curves, viz. residual protease activity offive proteases (two acid-stable proteases derived from Nocardiopsis, andthree reference proteases (Sub. Novo and Sub. Novo (Y217L), both derivedfrom Bacillus amyloliquefaciens, and SAVINASE™) after incubation for 2hours, at a temperature of 37° C., and at pH-values in the range of pH 2to pH 11; the activity is relative to residual activity after a 2 hourincubation at pH 9.0, and 5° C.

FIG. 2 shows pH-activity curves, viz. protease activity between pH 3 andpH 11, relative to the protease activity at pH-optimum, of the same fiveproteases.

FIG. 3 shows temperature-activity curves at pH 9.0, viz. proteaseactivity at pH 9.0 between 15° C. and 80° C., relative to proteaseactivity at the optimum temperature, of the same five proteases.

DETAILED DESCRIPTION OF THE INVENTION

The term protease as used herein is an enzyme that hydrolyzes peptidebonds (has protease activity). Proteases are also called e.g.peptidases, proteinases, peptide hydrolases, or proteolytic enzymes.

Preferred proteases for use according to the invention are of theendo-type that act internally in polypeptide chains (endopeptidases).Endopeptidases show activity on N- and C-terminally blocked peptidesubstrates that are relevant for the specificity of the protease inquestion.

Included in the above definition of protease are any enzymes belongingto the EC 3.4 enzyme group (including each of the thirteensub-subclasses thereof) of the EC list (Enzyme Nomenclature 1992 fromNC-IUBMB, 1992), as regularly supplemented and updated, see e.g. theWorld Wide Web (WWW) at www.chem.qmw.ac.uk/iubmb/enzyme/index.html.

Proteases are classified on the basis of their catalytic mechanism intothe following groupings: serine proteases (S), cysteine proteases (C),aspartic proteases (A), metalloproteases (M), and unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

The Nocardiopsis proteases disclosed herein are serine proteases.

In a particular embodiment the proteases for use according to theinvention are serine proteases. The term serine protease refers toserine peptidases and their clans as defined in the above Handbook. Inthe 1998 version of this handbook, serine peptidases and their clans aredealt with in chapters 1-175. Serine proteases may be defined aspeptidases in which the catalytic mechanism depends upon the hydroxylgroup of a serine residue acting as the nucleophile that attacks thepeptide bond. Examples of serine proteases for use according to theinvention are proteases of Clan SA, e.g. Family S2 (Streptogrisin), e.g.Sub-family S2A (alpha-lytic protease), as defined in the above Handbook.

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-values arepH 5, 6, 7, 8, 9, 10, or 11. Examples of assay-temperatures are 25, 30,35, 37, 40, 45, 50, 55, 60, 65, or 70° C.

Examples of protease substrates are casein, and pNA-substrates, such asSuc-AAPF-pNA (available e.g. from Sigma S-7388). The capital letters inthis pNA-substrate refers to the one-letter amino acid code. Anotherexample is Protazyme AK (azurine-dyed crosslinked casein prepared astablets by Megazyme T-PRAK). For pH-activity and pH-stability studies,the pNA-substrate is preferred, whereas for temperature-activitystudies, the Protazyme AK substrate is preferred.

Examples of protease assays are described in the experimental part.

There are no limitations on the origin of the protease for use accordingto the invention. Thus, the term protease includes not only natural orwild-type proteases, but also any mutants, variants, fragments etc.thereof exhibiting protease activity, as well as synthetic proteases,such as shuffled proteases, and consensus proteases. Such geneticallyengineered proteases can be prepared as is generally known in the art,e.g. by Site-directed Mutagenesis, by PCR (using a PCR fragmentcontaining the desired mutation as one of the primers in the PCRreactions), or by Random Mutagenesis. The preparation of consensusproteins is described in e.g. EP 897985.

Examples of acid-stable proteases for use according to the invention are

(i) the proteases derived from Nocardiopsis sp. NRRL 18262, andNocardiopsis alba;

(ii) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95%amino acid identity to any of the proteases of (i);

(iii) proteases of at least 60, 65, 70, 75, 80, 85, 90, or at least 95%identity to any of SEQ ID NO: 1, and/or SEQ ID NO: 2.

For calculating percentage identity, any computer program known in theart can be used. Examples of such computer programs are the Clustal Valgorithm (Higgins, D. G., and Sharp, P. M. (1989), Gene (Amsterdam),73, 237-244; and the GAP program provided in the GCG version 8 programpackage (Program Manual for the Wisconsin Package, Version 8, GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman,S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-453.

When using the Clustal V algorithm for calculating the percentage ofidentity between two protein sequences, a PAM250 residue weight table isused, together with the default settings of the MegAlign program, v4.03,in the Lasergene software package (DNASTAR Inc., 1228 South Park Street,Madison, Wis. 53715, US). Default settings for multiple alignments are agap penalty of 10 and a gap length penalty of 10. For calculatingpercentage identity between two protein sequences the following settingsare used: Ktuple of 1, gap penalty of 3, window of 5, and 5 diagonalssaved.

When using GAP, the following settings are applied for polypeptidesequence comparison: GAP creation penalty of 5.0 and GAP extensionpenalty of 0.3.

In a particular embodiment, the protease for use according to theinvention is a microbial protease, the term microbial indicating thatthe protease is derived from, or originates from, a microorganism, or isan analogue, a fragment, a variant, a mutant, or a synthetic proteasederived from a microorganism. It may be produced or expressed in theoriginal wild-type microbial strain, in another microbial strain, or ina plant; i.e. the term covers the expression of wild-type, naturallyoccurring proteases, as well as expression in any host of recombinant,genetically engineered or synthetic proteases.

The term microorganism as used herein includes Archaea, bacteria, fungi,vira etc.

Examples of microorganisms are bacteria, e.g. bacteria of the phylumActinobacteria phy.nov., e.g. of class I: Actinobacteria, e.g. of theSubclass V: Actinobacteridae, e.g. of the Order I: Actinomycetales, e.g.of the Suborder XII: Streptosporangineae, e.g. of the Family II:Nocardiopsaceae, e.g. of the Genus I: Nocardiopsis, e.g. Nocardiopsissp. NRRL 18262, and Nocardiopsis alba; or mutants or variants thereofexhibiting protease activity. This taxonomy is on the basis of Bergey'sManual of Systematic Bacteriology, 2^(nd) edition, 2000, Springer(preprint: Road Map to Bergey's).

Further examples of microorganisms are fungi, such as yeast orfilamentous fungi.

In another embodiment the protease is a plant protease. An example of aprotease of plant origin is the protease from the sarcocarp of melonfruit (Kaneda et al, J. Biochem. 78, 1287-1296 (1975).

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants, such as cows, sheep andhorses. In a particular embodiment, the animal is a non-ruminant animal.Non-ruminant animals include mono-gastric animals, e.g. pigs or swine(including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys and chicken (including but not limited tobroiler chicks, layers); young calves; and fish (including but notlimited to salmon).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the protease can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In the present context, the term acid-stable means, that the proteaseactivity of the pure protease enzyme, in a dilution corresponding toA₂₈₀=1.0, and following incubation for 2 hours at 37° C. in thefollowing buffer:

100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mMCaCl₂, 150 mM KCl, 0.01% Triton® X-100, pH 3.5, is at least 40% of thereference activity, as measured using the assay described in Example 2Cherein (substrate: Suc-AAPF-pNA, pH 9.0, 25° C.).

In particular embodiments of the above acid-stability definition, theprotease activity is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, or at least 97% of the reference activity.

The term reference activity refers to the protease activity of the sameprotease, following incubation in pure form, in a dilution correspondingto A₂₈₀=1.0, for 2 hours at 5° C. in the following buffer: 100 mMsuccinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl₂, 150mM KCl, 0.01% Triton® X-100, pH 9.0, wherein the activity is determinedas described above.

In other words, the method of determining acid-stability comprises thefollowing steps:

a) The protease sample to be tested (in pure form, A₂₅₀=1.0) is dividedin two aliquots (I and II);

b) Aliquot I is incubated for 2 hours at 37° C. and pH 3.5;

c) Residual activity of aliquot I is measured (pH 9.0 and 25° C.);

d) Aliquot II is incubated for 2 hours at 5° C. and pH 9.0;

e) Residual activity of aliquot II is measured (pH 9.0 and 25° C.);

f) Percentage residual activity of aliquot I relative to residualactivity of aliquot II is calculated.

Alternatively, in the above definition of acid-stability, the step b)buffer pH-value may be 1.0, 1.5, 2.0, 2.5, 3.0, 3.1, 3.2, 3.3, or 3.4.

In other alternative embodiments of the above acid-stability definitionrelating to the above alternative step b) buffer pH-values, the residualprotease activity as compared to the reference, is at least 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or atleast 97%.

In alternative embodiments, pH values of 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5can be applied for the step d) buffer.

In the above acid-stability definition, the term A₂₈₀=1.0 means suchconcentration (dilution) of said pure protease which gives rise to anabsorption of 1.0 at 280 nm in a 1 cm path length cuvette relative to abuffer blank.

And in the above acid-stability definition, the term pure proteaserefers to a sample with a A₂₈₀/A₂₆₀ ratio above or equal to 1.70 (seeExample 2E), and which by a scan of a Coomassie-stained SDS-PAGE gel ismeasured to have at least 95% of its scan intensity in the bandcorresponding to said protease (see Example 2A). In the alternative, theA₂₈₀/A₂₆₀ ratio is above or equal to 1.50, 1.60, 1.65, 1.70, 1.75, 1.80,1.85, or above or equal to 1.90.

However, for the uses according to the invention, the protease need notbe that pure; it may e.g. include other enzymes, even other proteases,in which case it could be termed a protease preparation. Nevertheless, awell-defined protease preparation is advantageous. For instance, it ismuch easier to dose correctly to the feed a protease that is essentiallyfree from interfering or contaminating other proteases. The term dosecorrectly refers in particular to the objective of obtaining consistentand constant results, and the capability of optimising dosage based uponthe desired effect.

In a particular embodiment, the protease, in the form in which it isadded to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the protease preparation is atleast 50% pure as determined by Size-exclusion chromatography (seeExample 8).

In other particular embodiments the protease preparation is at least 60,70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by thismethod.

In the alternative, the term well-defined means, that a fractionation ofthe protease preparation on an appropriate Size-exclusion column revealsonly one major protease component.

The skilled worker will know how to select an appropriate Size-exclusionchromatography column. He might start by fractionating the preparationon e.g. a HiLoad26/60 Superdex75pg column from Amersham PharmaciaBiotech (see Example 8). If the peaks would not be clearly separated hewould try different columns (e.g. with an amended column particle sizeand/or column length), and/or he would amend the sample volume. Bysimple and common trial-and-error methods he would thereby arrive at acolumn with a sufficient resolution (clear separation of peaks), on thebasis of which the purity calculation is performed as described inExample 8.

The protease preparation can be (a) added directly to the feed (or useddirectly in the treatment process of vegetable proteins), or (b) it canbe used in the production of one or more intermediate compositions suchas feed additives or premixes that is subsequently added to the feed (orused in a treatment process). The degree of purity described aboverefers to the purity of the original protease preparation, whether usedaccording to (a) or (b) above.

Protease preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the protease is produced by traditionalfermentation methods.

Such protease preparation may of course be mixed with other enzymes.

In one particular embodiment, the protease for use according to theinvention, besides being acid-stable, also has a pH-activity optimumclose to neutral.

The term pH-activity optimum close to neutral means one or more of thefollowing: That the pH-optimum is in the interval of pH 6.0-11.0, or pH7.0-11.0, or pH 6.0-10.0, or pH 7.0-10.0, or pH 8.0-11.0, or pH 8.0-10.0(see Example 2B and FIG. 2 herein).

In another particular embodiment, the protease for use according to theinvention, besides being acid-stable, is also thermostable.

The term thermostable means one or more of the following: That thetemperature optimum is at least 50° C., 52° C., 54° C., 56° C., 58° C.,60° C., 62° C., 64° C., 66° C., 68° C., or at least 70° C., referencebeing made to Example 2D and FIG. 3 herein.

In a further particular embodiment, the protease for use according tothe invention is capable of solubilizing vegetable proteins according tothe in vitro model of Example 4 herein.

The term vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g. soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, and sorghum.

The treatment according to the invention of vegetable proteins with atleast one acid-stable protease results in an increased solubilization ofvegetable proteins.

The following are examples of % solubilized protein obtainable using theproteases of the invention: At least 74.0%, 74.5%, 75.0%, 75.5%, 76.0%,76.5%, 77.0%, or at least 77.5%, reference being had to the in vitromodel of Example 4 herein.

The term solubilization of proteins basically means bringing protein(s)into solution. Such solubilization may be due to protease-mediatedrelease of protein from other components of the usually complex naturalcompositions such as feed. Solubilization can be measured as an increasein the amount of soluble proteins, by reference to a sample with noprotease treatment (see Example 4 herein).

In a particular embodiment of a treatment process the protease(s) inquestion is affecting (or acting on, or exerting its solubilizinginfluence on the vegetable proteins or protein sources. To achieve this,the vegetable protein or protein source is typically suspended in asolvent, e.g. an aqueous solvent such as water, and the pH andtemperature values are adjusted paying due regard to the characteristicsof the enzyme in question. For example, the treatment may take place ata pH-value at which the relative activity of the actual protease is atleast 50, or 60, or 70, or 80 or 90%. Likewise, for example, thetreatment may take place at a temperature at which the relative activityof the actual protease is at least 50, or 60, or 70, or 80 or 90% (theserelative activities being defined as in Example 2 herein). The enzymaticreaction is continued until the desired result is achieved, followingwhich it may or may not be stopped by inactivating the enzyme, e.g. by aheat-treatment step.

In another particular embodiment of a treatment process of theinvention, the protease action is sustained, meaning e.g. that theprotease is added to the vegetable proteins or protein sources, but itssolubilizing influence is so to speak not switched on until later whendesired, once suitable solubilizing conditions are established, or onceany enzyme inhibitors are inactivated, or whatever other means couldhave been applied to postpone the action of the enzyme.

In one embodiment the treatment is a pre-treatment of animal feed orvegetable proteins for use in animal feed, i.e. the proteins aresolubilized before intake.

The term improving the nutritional value of an animal feed meansimproving the availability of the proteins, thereby leading to increasedprotein extraction, higher protein yields, and/or improved proteinutilisation. The nutritional value of the feed is therefore increased,and the growth rate and/or weight gain and/or feed conversion (i.e. theweight of ingested feed relative to weight gain) of the animal is/areimproved.

The protease can be added to the feed in any form, be it as a relativelypure protease, or in admixture with other components intended foraddition to animal feed, i.e. in the form of animal feed additives, suchas the so-called pre-mixes for animal feed.

Animal Feed Additives

Apart from the acid-stable protease, the animal feed additives of theinvention contain at least one fat-soluble vitamin, and/or at least onewater-soluble vitamin, and/or at least one trace mineral, and/or atleast one macro mineral.

Further, optional, feed-additive ingredients are coloring agents, aromacompounds, stabilisers, and/or at least one other enzyme selected fromamongst phytases EC 3.1.3.8 or 3.1.3.26; xylanases EC 3.2.1.8;galactanases EC 3.2.1.89; and/or beta-glucanases EC 3.2.1.4 (EC refersto Enzyme Classes according to Enzyme Nomenclature 1992 from NC-IUBMB,1992), see also the World Wide Web (WWW) atwww.chem.qmw.ac.uk/iubmb/enzyme/index.html.

In a particular embodiment these other enzymes are well-defined (asdefined and exemplified above for protease preparations, i.a. byreference to Example 8).

Usually fat—and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with an acid-stable protease accordingto the invention, is an animal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01-10.0%; more particularly0.05-5.0%; or 0.2-1.0% (% meaning g additive per 100 g feed). This is soin particular for premixes.

Accordingly, the concentrations of the individual components of theanimal feed additive, e.g. the premix, can be found by multiplying thefinal in-feed concentration of the same component by, respectively,10-10000; 20-2000; or 100-500 (referring to the above three percentageinclusion intervals).

Guidelines for desired final concentrations, i.e.in-feed-concentrations, of such individual feed and feed additivecomponents are indicated in Table A below.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g. vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g. Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components—exemplified withpoultry and piglets/pigs—are listed in Table A below. Nutritionalrequirement means that these components should be provided in the dietin the concentrations indicated. These data are compiled from:

NRC, Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C. 1988; and

NRC, Nutrient requirements of poultry, ninth revised edition 1994,subcommittee on poultry nutrition, committee on animal nutrition, boardof agriculture, national research council. National Academy Press,Washington, D.C. 1994.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A. At leastone means either of, one or more of, one, or two, or three, or four andso forth up to all thirteen, or up to all fifteen individual components.

More specifically, this at least one individual component is included inthe additive of the invention in such an amount as to provide anin-feed-concentration within the range indicated in column four, orcolumn five, or column six of Table A.

As explained above, corresponding feed additive concentrations can befound by multiplying the interval limits of these ranges with 10-10000;20-2000; or 100-500. As an example, considering which premix-content ofvitamin A would correspond to the feed-content of 10-10000 IU/kg, thisexercise would lead to the following intervals: 100-10⁸ IU; or 200-2×10⁷IU; or 1000-5×10⁸ IU per kg additive.

TABLE A Nutrient requirements - and preferred ranges Nutrients providedper kg Piglets/Pigs/ diet Poultry Sows Range 1 Range 2 Range 3Fat-soluble vitamins Vitamin A/[IU] −5000 1300-4000   10-10000  50-8000 100-6000 Vitamin D₃/[IU] −1100 150-200   2-3000   5-2000  10-1500Vitamin E/[IU] −12 11-22 0.02-100  0.2-80  0.5-50  Vitamin K/[mg]0.5-1.5 −0.5  0.005-10.0  0.05-5.0  0.1-3.0 Water-soluble vitaminsB₁₂/[mg] −0.003 0.005-0.02  0.0001-1.000  0.0005-0.500  0.001-0.100Biotin/[mg] 0.100-0.25  0.05-0.08 0.001-10.00 0.005-5.00  0.01-1.00Choline/[mg]  800-1600 300-600   1-10000   5-5000  10-3000 Traceminerals Manganese/[mg] −60 2.0-4.0   0.1-1000  0.5-500  1.0-100Zinc/[mg] 40-70  50-100   1-1000  5-500  10-300 Iron/[mg] 50-80  40-100  1-1000  5-500  10-300 Copper/[mg] 6-8 3.0-6.0   0.1-1000  0.5-1001.0-25  Iodine/[mg] −0.4 −0.14 0.01-100  0.05-10   0.1-1.0 Selenium/[mg]−0.2 0.10-0.30 0.005-100   0.01-10.0 0.05-1.0  Macro mineralsCalcium/[g]  8-40 5-9  0.1-200  0.5-150  1-100 Phosphorus, as 3-61.5-6    0.1-200  0.5-150  1-50 available phosphorus/[g]Animal Feed Compositions

Animal feed compositions or diets have a relatively high content ofprotein. According to the National Research Council (NRC) publicationsreferred to above, poultry and pig diets can be characterised asindicated in Table B below, columns 2-3. Fish diets can be characterisedas indicated in column 4 of Table B. Furthermore such fish diets usuallyhave a crude fat content of 200-310 g/kg. These fish diet areexemplified with diets for Salmonids and designed on the basis ofAquaculture, principles and practices, ed. T. V. R. Pillay, BlackwellScientific Publications Ltd. 1990; Fish nutrition, second edition, ed.John E. Halver, Academic Press Inc. 1989.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneprotease as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B below(R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25 as stated in Animal Nutrition,4th edition, Chapter 13 (Eds. P. McDonald, R. A. Edwards and J. F. D.Greenhalgh, Longman Scientific and Technical, 1988, ISBN 0-582-40903-9).The nitrogen content is determined by the Kjeldahl method (A.O.A.C.,1984, Official Methods of Analysis 14th ed., Association of OfficialAnalytical Chemists, Washington D.C.).

Metabolizable energy can be calculated on the basis of the NRCpublication Nutrient Requirements of Swine (1988) pp. 2-6, and theEuropean Table of Energy Values for Poultry Feedstuffs, Spelderholtcentre for poultry research and extension, 7361 DA Beekbergen, TheNetherlands. Grafisch bedrijf Ponsen & Iooijen by, Wageningen. ISBN90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein or protein source as definedabove.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-10% fish meal; and/or 0-20% whey.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) orpelleted feed. Typically, the milled feedstuffs are mixed and sufficientamounts of essential vitamins and minerals are added according to thespecifications for the species in question. Enzymes can be added assolid or liquid enzyme formulations. For example, a solid enzymeformulation is typically added before or during the mixing step; and aliquid enzyme preparation is typically added after the pelleting step.The enzyme may also be incorporated in a feed additive or premix. Thefinal enzyme concentration in the diet is within the range of 0.01-200mg enzyme protein per kg diet, for example in the range of 5-30 mgenzyme protein per kg animal diet.

Examples of animal feed compositions are shown in Example 7.

TABLE B Range values for energy, protein and minerals in animal dietsPiglets/Pigs/ Poultry Sows Fish Nutrient Min-Max Min-Max Min-Max R. 1 R.2 R. 3 R. 4 R. 5 Metabolizable 12.1-13.4 12.9-13.5 14-25 10-30 11-2811-26 12-25 energy, MJ/kg Crude 124-280 120-240 300-480  50-800  75-700100-600 110-500 120-490 protein, g/kg Calcium,  8-40 5-9 10-15  0.1-200 0.5-150  1-100  4-50 g/kg Available 2.1-6.0 1.5-5.5  3-12  0.1-200 0.5-150  1-100  1-50  1-25 Phosphorus, g/kg Methionine, 3.2-5.5 — 12-16 0.1-100 0.5-75   1-50  1-30 g/kg Methionine 4-9 2.3-6.8 —  0.1-150 0.5-125  1-80 plus Cysteine, g/kg Lysine, g/kg 2.5-11   6-14 12-220.5-50  0.5-40   1-30

In particular embodiments of the method of the invention for treatingvegetable proteins, a further step of adding phytase is also included.And in further particular embodiments, in addition to the combinedtreatment with phytase and protease, further enzymes may also be added,wherein these enzymes are selected from the group comprising otherproteases, phytases, lipolytic enzymes, and glucosidase/carbohydraseenzymes. Examples of such enzymes are indicated in WO 95/28850.

The protease should of course be applied in an effective amount, i.e. inan amount adequate for improving solubilization and/or improvingnutritional value of feed. It is at present contemplated that the enzymeis administered in one or more of the following amounts (dosage ranges):0.01-200; or 0.01-100; or 0.05-1.00; or 0.05-50; or 0.10-10—all theseranges being in mg protease protein per kg feed (ppm).

For determining mg protease protein per kg feed, the protease ispurified from the feed composition, and the specific activity of thepurified protease is determined using a relevant assay (see underprotease activity, substrates, and assays). The protease activity of thefeed composition as such is also determined using the same assay, and onthe basis of these two determinations, the dosage in mg protease proteinper kg feed is calculated.

The same principles apply for determining mg protease protein in feedadditives.

Of course, if a sample is available of the protease used for preparingthe feed additive or the feed, the specific activity is determined fromthis sample (no need to purify the protease from the feed composition orthe additive).

Many vegetables contain anti-nutritional factors such as lectins andtrypsin inhibitors. The most important anti-nutritional factors ofsoybean are the lectin soybean agglutinin (SBA), and the soybean trypsininhibitor (STI).

Lectins are proteins that bind to specific carbohydrate-containingmolecules with considerable specificity, and when ingested they becomebound to the intestinal epithelium. This may lead to reduced viabilityof the epithelial cells and reduced absorption of nutrients.

SBA is a glycosylated, tetrameric lectin with a subunit molecular weightof about 30 kDa and a high affinity for N-acetylgalactosamine.

Trypsin inhibitors affect the intestinal proteolysis reducing proteindigestibility, and also increase the secretion of digestive enzymes fromthe pancreas leading to a loss of amino acids in the form of digestiveenzymes. An example of a trypsin inhibitor is the Bowman-Birk Inhibitor,which has a molecular weight of about 8 kDa, contains 7 disulfidebridges and has two inhibitory loops specific for trypsin-like andchymotrypsin-like proteases. Other examples are the so-called KunitzInhibitors of Factors (e.g. the Soybean Kunitz Trypsin Inhibitor thatcontains one binding site for trypsin-like proteases and has a molecularweight of about 20 kDa).

The proteases for use according to the invention have been shown tohydrolyze anti-nutritional factors like SBA lectin, and the trypsininhibitors Bowman Birk Inhibitor and The Soybean Kunitz Factor. See theexperimental part, Example 5.

Thus, the invention also relates to the use of acid-stable proteases forhydrolysing, or reducing the amount of, anti-nutritional factors, e.g.SBA lectin, and trypsin inhibitors, such as the Bowman Birk Inhibitor,and Kunitz Factors, such as the Soybean Kunitz Factor.

EXAMPLE 1 Screening for Acid-Stable Proteases

A large number of proteases were analyzed for stability at pH 3, withthe objective of identifying proteases that have the necessary stabilityto pass through the acidic stomach of mono-gastric animals.

The proteases had been purified by conventional chromatographic methodssuch as ion-exchange chromatography, hydrophobic interactionchromatography and size exclusion chromatography (see e.g. ProteinPurification, Principles, High Resolution Methods, and Applications.Editors: Jan-Christer Janson, Lars Rydén, VCH Publishers, 1989).

Protease activity was determined as follows: The protease was incubatedwith 1.67% Hammarsten casein at 25° C., pH 9.5 for 30 minutes, then TCA(tri-chloro acetic acid) was added to a final concentration of 2% (w/w),the mixture was filtrated to remove the sediment, and the filtrate wasanalyzed for free primary amino groups (determined in a colometric assaybased on OPA (o-phthal-dialdehyde) by measuring the absorbance at 340nm, using a serine standard (Biochemische Taschenbuch teil II,Springer-Verlag (1964), p. 93 and p. 102). One Casein Protease Unit(CPU) is defined as the amount of enzyme liberating 1 mmol ofTCA-soluble primary amino groups per minute under standard conditions,i.e. 25° C. and pH 9.5.

The proteases were diluted to an activity of 0.6 CPU/I in water, dividedin two aliquots and each aliquot was then further diluted to 0.3 CPU/Iwith 100 mM citrate buffer, pH 3, and 100 mM phosphate buffer, pH 7respectively. The diluted samples were incubated at 37° C. for 1 hour,and 20 microliters of the samples were applied to holes in 1% agaroseplates containing 1% skim milk. The plates (pH 7.0) were incubated at37° C. over night and clearing zones were measured.

A number of proteases performed well in this test, and the following twohave now been characterised: The Nocardiopsis alba and Nocardiopsis sp.NRRL 18262 proteases described in Example 2. The strain of Nocardiopsisalba has been deposited according to the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure at DSMZ-Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH, Mascheroder Weg 1b, D-38124Braunschweig, Germany, as follows:

Deposit date: 22 Jan. 2001

CBS No.: Nocardiopsis alba DSM 14010

The deposit was made by Novozymes A/S and was later assigned toHoffmann-La Roche AG.

EXAMPLE 2 Preparation, Characterization and Comparative Study ofNocardiopsis Proteases Fermentation

Nocardiopsis alba was inoculated from tryptone yeast agar plates intoshake flasks each containing 100 ml HG-23 medium with the followingcomposition: Oatmeal 45 g/l, Yeast Extract 2 g/l, di-sodium hydrogenphosphate 12 g/l, Potassium di-hydrogen phosphate 6 g/l, Pluronic PE6100 0.2 ml/l in distilled water. The strain was fermented for 9 days at37 degree C.

Purification

The culture broth was centrifuged at 10000×g for 30 minutes in 1 literbeakers. The supernatants were combined and further clarified by afiltration though a Seitz K-250 depth filter plate. The clear filtratewas concentrated by ultrafiltration on a 3 kDa cut-off polyether sulfonecassette (Filtron). The concentrated enzyme was transferred to 50 mMH₃BO₃, 5 mM 3,3′-dimethyl glutaric acid, 1 mM CaCl₂, pH 7 (Buffer A) ona G25 Sephadex column (Amersham Pharmacia Biotech), and applied to aBacitracin agarose column (Upfront Chromatography A/S) equilibrated inBuffer A. After washing the Bacitracin column with Buffer A to removeunbound protein, the protease was eluted from the column using Buffer Asupplemented with 25% 2-propanol and 1 M sodium chloride. The fractionswith protease activity were pooled and transferred to 20 mMCH₃COOH/NaOH, 1 mM CaCl₂, pH 4.5 (Buffer B) by chromatography on a G25Sephadex column (Amersham Pharmacia Biotech). The buffer exchangedprotease pool was applied to a SOURCE 30S column (Amersham PharmaciaBiotech) equilibrated in Buffer B. After washing the SOURCE 30S columnwith Buffer B, the protease was eluted with an increasing linear NaClgradient (0 to 0.25 M) in Buffer B. Fractions from the column weretested for protease activity and protease containing fractions wereanalyzed by SDS-PAGE. Pure fractions were pooled and used for furthercharacterization.

The protease of Nocardiopsis sp. NRRL 18262 was prepared usingconventional methods, as generally described above for the protease ofNocardiopsis.

Characterization

The protease derived from Nocardiopsis alba was found to have amolecular weight of Mr=21 kDa (SDS-PAGE), and the following partial(N-terminal (MVS)) amino acid sequence was determined:

ADIIGGLAYTMGGRCSV. (SEQ ID NO: 2)

The protease derived from Nocardiopsis sp. NRRL 18262 has the followingsequence of 188 amino acids:

(SEQ ID NO: 1) ADIIGGLAYTMGGRCSVGFAATNAAGQPGFVTAGHCGRVGTQVTIGNGRGVFEQSVFPGNDAAFVRGTSNFTLTNLVSRYNTGGYAAVAGHNQAPIGSSVCRSGSTTGWHCGTIQARGQSVSYPEGTVTNMTRTTVCAEPGDSGGSYISGTQAQGVTSGGSGNCRTGGTTFYQEVTPMVNSWGVRLRT.

The purpose of this characterisation was to study their pH-stability,pH-activity and temperature-activity profiles, in comparison to Sub.Novo, Sub. Novo (Y217L), and SAVINASE™.

Sub. Novo is subtilisin from Bacillus amyloliquefaciens, and Sub. Novo(Y217L) is the mutant thereof that is disclosed in WO 96/05739. Sub.Novo was prepared and purified from a culture of the wild-type strainusing conventional methods, whereas the mutant was prepared as describedin Examples 1-2, and 15-16 of EP 130756.

SAVINASE™ is a subtilisin derived from Bacillus clausii (previouslyBacillus lentus NCIB 10309), commercially available from Novozymes A/S,Krogshoejvej, DK-2880 Bagsvaerd, Denmark. Its preparation is describedin U.S. Pat. No. 3,723,250.

EXAMPLE 2A Determination of SDS-PAGE Purity of Protease Samples

The SDS-PAGE purity of the protease samples was determined by thefollowing procedure:

40 microliters protease solution (A₂₈₀ concentration=0.025) was mixedwith 10 microliters 50% (w/v) TCA (trichloroacetic acid) in an Eppendorftube on ice. After half an hour on ice the tube was centrifuged (5minutes, 0° C., 14.000×g) and the supernatant was carefully removed. 20microliters SDS-PAGE sample buffer (200 microliters Tris-Glycine SDSSample Buffer (2×) (125 mM Tris/HCl, pH 6.8, 4% (w/v) SDS, 50 ppmbromophenol blue, 20% (v/v) Glycerol, LC2676 from NOVEX™)+160microliters dist. water+20 microliters beta-mercaptoethanol+20microliters 3 M unbuffered Tris Base (Sigma T-1503) was added to theprecipitate and the tube was boiled for 3 minutes. The tube wascentrifuged shortly and 10 microliter sample was applied to a 4-20%gradient Tris-Glycine precast gel from NOVEX™ (polyacrylamide gradientgel based on the Laemmli chemistry but without SDS in the gel, (Laemmli,U. K., (1970) Nature, vol. 227, pp. 680-685), EC60255). Theelectrophoresis was performed with Tris-Glycine running buffer (2.9 gTris Base, 14.4 g Glycine, 1.0 g SDS, distilled water to 1 liter) inboth buffer reservoirs at a 150V constant voltage until the bromophenolblue tracking dye had reached the bottom of the gel. Afterelectrophoresis, the gel was rinsed 3 times, 5 minutes each, with 100 mlof distilled water by gentle shaking. The gel was then gently shakedwith Gelcode® Blue Stain Reagent (colloidal Comassie G-250 product fromPIERCE, PIERCE cat. No. 24592) for one hour and washed by gentle shakingfor 8 to 16 hours with distilled water with several changes of distilledwater. Finally, the gel was dried between 2 pieces of cellophane. Driedgels were scanned with an Arcus II scanner from AGFA equipped withFotolook 95 v2.08 software and imported to the image evaluation softwareCREAM™ for Windows (catalogue nos. 990001 and 990005, Kem-En-Tec,Denmark) by the File/Acquire command with the following settings (ofFotolook 95 v2.08): Original=Reflective, Mode=Color RGB, Scanresolution=240 ppi, Output resolution=120 lpi, Scale factor=100%,Range=Histogram with Global selection and Min=0 and Max=215,ToneCurve=None, Sharpness=None, Descreen=None and Flavor=None, therebyproducing an *.img picture file of the SDS-PAGE gel, which was used forevaluation in CREAM™. The *.img picture file was evaluated with the menucommand Analysis/1-D. Two scan lines were placed on the *.img picturefile with the Lane Place Tool: A Sample scan line and a Background scanline. The Sample scan line was placed in the middle of a sample lane(with the protease in question) from just below the application slot tojust above the position of the Bromphenol blue tracking dye. TheBackground scan line was placed parallel to the Sample scan line, but ata position in the pictured SDS-PAGE gel where no sample was applied,start and endpoints for the Background scan line were perpendicular tothe start and endpoints of the Sample scan line. The Background scanline represents the true background of the gel. The width and shape ofthe scan lines were not adjusted. The intensity along the scan lineswhere now recorded with the 1-D/Scan menu command with Mediumsensitivity. Using the 1-D/Editor menu command, the Background scan wassubtracted from the Sample scan. Then the 1-D/Results menu command wasselected and the Area % of the protease peak, as calculated by theCREAM™ software, was used as the SDS-PAGE purity of the proteases.

All the protease samples had an SDS-PAGE purity of above 95%.

EXAMPLE 2B pH-Activity Assay

Suc-AAPF-pNA (Sigma® S-7388) was used for obtaining pH-activityprofiles.

Assay buffer: 100 mM succinic acid (Merck 1.00682), 100 mM HEPES (SigmaH-3375), 100 mM CHES (Sigma C-2885), 100 mM CABS (Sigma C-5580), 1 mMCaCl₂, 150 mM KCl, 0.01% Triton® X-100, adjusted to pH-values 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or 11.0 with HCl or NaOH.

Assay temperature; 25° C.

A 300 microliter protease sample (diluted in 0.01% Triton® X-100) wasmixed with 1.5 ml of the assay buffer at the respective pH value,bringing the pH of the mixture to the pH of the assay buffer. Thereaction was started by adding 1.5 ml pNA substrate (50 mg dissolved in1.0 ml DMSO and further diluted 45× with 0.01% Triton® X-100) and, aftermixing, the increase in A₄₀₅ was monitored by a spectrophotometer as ameasurement of the protease activity at the pH in question. The assaywas repeated with the assay buffer at the other pH values, and theactivity measurements were plotted as relative activity against pH. Therelative activities were normalized with the highest activity(pH-optimum), i.e. setting activity at pH-optimum to 1, or to 100%. Theprotease samples were diluted to ensure that all activity measurementsfell within the linear part of the dose-response curve for the assay.

EXAMPLE 2C pH-Stability Assay

Suc-AAPF-pNA (Sigma® S-7388) was used for obtaining pH-stabilityprofiles.

Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mMCABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton® X-100 adjusted to pH-values2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 or 11.0 withHCl or NaOH.

Each protease sample (in 1 mM succinic acid, 2 mM CaCl₂, 100 mM NaCl, pH6.0 and with an A₂₈₀ absorption>10) was diluted in the assay buffer ateach pH value tested to A₂₈₀=1.0. The diluted protease samples wereincubated for 2 hours at 37° C. After incubation, protease samples werediluted in 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS,1 mM CaCl₂, 150 mM KCl, 0.01% Triton® X-100, pH 9.0, bringing the pH ofall samples to pH 9.0.

In the following activity measurement, the temperature was 25° C.

300 microliters diluted protease sample was mixed with 1.5 ml of the pH9.0 assay buffer and the activity reaction was started by adding 1.5 mlpNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45×with 0.01% Triton® X-100) and, after mixing, the increase in A₄₀₅ wasmonitored by a spectrophotometer as a measurement of the (residual)protease activity. The 37° C. incubation was performed at the differentpH-values and the activity measurements were plotted as residualactivities against pH. The residual activities were normalized with theactivity of a parallel incubation (control), where the protease wasdiluted to A₂₈₀=1.0 in the assay buffer at pH 9.0 and incubated for 2hours at 5° C. before activity measurement as the other incubations. Theprotease samples were diluted prior to the activity measurement in orderto ensure that all activity measurements fell within the linear part ofthe dose-response curve for the assay.

EXAMPLE 2D Temperature-Activity Assay

Protazyme AK tablets were used for obtaining temperature profiles.Protazyme AK tablets are azurine dyed crosslinked casein prepared astablets by Megazyme.

Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mMCABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton® X-100 adjusted to pH 9.0with NaOH.

A Protazyme AK tablet was suspended in 2.0 ml 0.01% Triton® X-100 bygentle stirring. 500 microliters of this suspension and 500 microlitersassay buffer were mixed in an Eppendorf tube and placed on ice. 20microliters protease sample (diluted in 0.01% Triton X-100) was added.The assay was initiated by transferring the Eppendorf tube to anEppendorf thermomixer, which was set to the assay temperature. The tubewas incubated for 15 minutes on the Eppendorf thermomixer at its highestshaking rate. By transferring the tube back to the ice bath, the assayincubation was stopped. The tube was centrifuged in an ice-coldcentrifuge for a few minutes and the A₆₅₀ of the supernatant was read bya spectrophotometer. A buffer blind was included in the assay (insteadof enzyme). A₆₅₀(protease)−A₆₅₀(blind) was a measurement of proteaseactivity. The assay was performed at different temperatures and theactivity measurements were plotted as relative activities againstincubation temperature. The relative activities were normalized with thehighest activity (temperature optimum). The protease samples werediluted to ensure that all activity measurements fell within the nearlinear part of the dose-response curve for the assay.

An overview of the activity optima (pH- and temperature activity) isseen in Table 1. pH-stability, pH-activity and temperature-activityprofiles are seen in FIGS. 1-3, and a detailed comparison of thepH-stability data for the proteases at acidic pH-values is seen in Table2.

TABLE 1 pH- and temperature optima of various proteases pH-optimumTemperature-optimum at Protease (pNA-substrate) pH 9.0 (Protazyme AK)Nocardiopsis sp. NRRL 10 70° C. 18262 Nocardiopsis alba 11 70° C. Sub.Novo¹ 10 70° C. Sub. Novo (Y217L)² 9 70° C. SAVINASE ™³ 9 70° C.

TABLE 2 pH-stability of various proteases, between pH 2.0 and 5.0Protease pH 2.0 pH 2.5 pH 3.0 pH 3.5 pH 4.0 pH 4.5 pH 5.0 Nocardiopsissp. 0.779 1.000 1.029 0.983 0.991 1.019 1.004] NRRL 18262 Nocardiopsisalba 0.929 0.993 1.009 1.005 0.969 1.037 0.992 Sub. Novo 0.007 0.0030.000 0.000 0.024 0.784 0.942 Sub. Novo (Y217L) 0.000 0.000 0.002 0.0030.350 0.951 0.996 Savinase ® 0.001 0.001 0.001 0.003 0.338 0.929 0.992

EXAMPLE 2E Absorption Purity of Purified Protease Samples

Determination of A₂₈₀/A₂₆₀ Ratio

The A₂₈₀/A₂₆₀ ratio of purified protease samples was determined asfollows.

A₂₆₀ means the absorption of a protease sample at 260 nm in a 1 cm pathlength cuvette relative to a buffer blank. A₂₈₀ means the absorption ofthe same protease sample at 280 nm in a 1 cm path length cuvetterelative to a buffer blank.

Samples of the purified proteases from Example 2 were diluted in bufferuntil the A₂₈₀ reading of the spectrophotometer is within the linearpart of its response curve. The A₂₈₀/A₂₆₀ ratio was determined from thereadings: For Nocardiopsis sp. NRRL 18262 1.83, and for Nocardiopsisalba 1.75.

EXAMPLE 3 Ability of Protease Derived from Nocardiopsis sp. NRRL 18262to Degrade Insoluble Parts of Soy Bean Meal (SBM)

The protease from Nocardiopsis sp. NRRL 18262 was tested for its abilityto make the insoluble/indigestible parts of SBM accessible to digestiveenzymes and/or added exogeneous enzymes.

Its performance was compared to two aspartate proteases, Protease I andProtease II, prepared as described in WO 95/02044. This document alsodiscloses their use in feed. Protease I is an Aspergillopepsin II typeof protease, and Protease II an Aspergillopepsin I type of protease(both aspartate proteases, i.e. non-subtilisin proteases) fromAspergillus aculeatus (reference being made to Handbook of ProteolyticEnzymes referred to above).

The test substrate, the so-called soy remnant, was produced in a processwhich mimics the digestive tract of mono-gastric animals, including apepsin treatment at pH 2, and a pancreatin treatment at pH 7.

In the pancreatin treatment step a range of commercial enzymes was addedin high dosages in order to degrade the SBM components that areaccessible to existing commercial enzymes.

The following enzymes, all commercially available from Novozymes NS,Denmark, were added: ALCALASE™ 2.4L, NEUTRASE™ 0.5L, FLAVOURZYME™ 1000L,ENERGEX™ L, BIOFEED™ Plus L, PHYTASE NOVO™ L. The SBM used was astandard 48% protein SBM for feed, which had been pelletized.

After the treatment only 5% of the total protein was left in theresulting soy remnant.

FITC Labelling Protocol

The remnant was subsequently labelled with FITC (Molecular Probes,F-143) as follows: Soy remnant (25 g wet, ˜5 g dry) was suspended in 100ml 0.1 M carbonate buffer, pH 9 and stirred 1 hour at 40° C. Thesuspension was cooled to room temperature and treated with fluorescein5-isothiocyanate (FITC) over night in the dark. Non-coupled probe wasremoved by ultrafiltration (10.000 Mw cut-off).

FITC-Assay

The FITC-labelled soy remnant was used for testing the ability of theproteases to degrade the soy remnant using the following assay: 0.4 mlprotease sample (with A₂₈₀=0.1) was mixed with 0.4 ml FITC-soy remnant(suspension of 10 mg/ml in 0.2 M sodium-phosphate buffer pH 6.5) at 37°C., and the relative fluorescence units (RFU 485/535 nm;excitation/monitoring wave length) measured after 0 hours, and after 22hours incubation. Before determination of the RFU, samples werecentrifuged for 1 min at 20.000×G and 250 microliter supernatant wastransferred to a black micro-titer tray. Measurements were performedusing a VICTOR 1420 Multilabel counter (In vitro, Denmark). RFU isgenerally described by lain D. Johnson in: Introduction to FluorescenceTechniques, Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes, Richard P. Haugland, 6^(th) edition, 1996 (ISBN0-9652240-0-7).

A blind sample was prepared by adding 0.4 ml buffer instead of enzymesample. RFU_(sample)=ΔRFU_(sample)−ΔRFU_(blind), where ΔRFU=RFU(22hours)−RFU(0 hours)

The resulting FITC values (RFU_(sample) values) are shown in Table 3below. The FITC values are generally with an error margin of +/−20.000.Contrary to Protease I and Protease II, the protease derived fromNocardiopsis sp. NRRL 18262 degraded the soy remnant to a significantextent.

TABLE 3 Ability of proteases to degrade soy remnant Protease FITC(+/−20000) Nocardiopsis sp. NRRL 18262 92900 Protease I −9200 ProteaseII −1200

EXAMPLE 4 In Vitro Testing of the Protease Derived from Nocardiopsis sp.NRRL 18262

The protease derived from Nocardiopsis sp. NRRL 18262 was tested,together with a protease derived from Bacillus sp. NCIMB 40484 (“PD498,”prepared as described in Example 1 of WO 93/24623), and together withFLAVOURZYME™, a protease-containing enzyme preparation from Aspergillusoryzae (commercially available from Novozymes NS, Bagsvaerd, Denmark),for its ability to solubilize maize-SBM (maize-Soy Bean Meal) proteinsin an in vitro digestion system (simulating digestion in monogastricanimals). For the blank treatments, maize-SBM was incubated in theabsence of exogenous proteases.

Outline of in vitro model Time Components added to flask course (min) 10g maize-SBM (60:40) + HCl/pepsin t = 0 (3000 U/g diet) + protease (0.1mg enzyme protein/g diet or 3.3 mg FLAVOURZYME ™/g diet), T = 40° C., pH= 3.0 NaOH, T = 40° C., pH 6 t = 60 NaHCO₃/pancreatin (8.0 mg/g diet), T= 40° C., pH 6-7 t = 80 Stop incubation and take samples, T = 0° C. t =320Substrates

10 g maize-SBM diet with a ratio maize-SBM of 6:4 (w/w) was used. Theprotein content was 43% (w/w) in SBM and 8.2% (w/w) in maize meal. Thetotal amount of protein in 10 g maize-SBM diet was 2.21 g.

Digestive Enzymes

Pepsin (Sigma P-7000; 539 U/mg, solid), pancreatin (Sigma P-7545;8xU.S.P. (US Pharmacopeia)).

Enzyme Protein Determinations

The amount of protease enzyme protein is calculated on the basis of theA₂₈₀ values and the amino acid sequences (amino acid compositions) usingthe principles outlined in S. C. Gill & P. H. von Hippel, AnalyticalBiochemistry 182, 319-326, (1989).

Experimental Procedure for In Vitro Model

1.10 g of substrate is weighed into a 100 ml flask.

2. At time 0 min, 46 ml HCl (0.1 M) containing pepsin (3000 U/g diet)and 1 ml of protease (0.1 mg enzyme protein/g diet, except forFLAVOURZYME™: 3.3 mg/g diet) are added to the flask while mixing. Theflask is incubated at 40° C.

3. At time 20-25 min, pH is measured.

4. At time 45 min, 16 ml of H₂O is added.

5. At time 60 min, 7 ml of NaOH (0.4 M) is added.

6. At time 80 min, 5 ml of NaHCO₃ (1 M) containing pancreatin (8.0 mg/gdiet) is added.

7. At time 90 min, pH is measured.

8. At time 300 min, pH is measured.

9. At time 320 min, aliquots of 30 ml are removed and centrifuged(10000×g, 10 min, 0° C.).

10. Total soluble protein in supernatants is determined.

Protein Determination

Supernatants are analyzed for protein content using the Kjeldahl method(determination of % nitrogen; A.O.A.C. (1984) Official Methods ofAnalysis 14^(th) ed. Association of Official Analytical Chemists,Washington D.C.).

Calculations

For all samples, in vitro protein solubility was calculated using theequations below.

Amount of protein in diet sample: 22.1% of 10 g=2.21 g

If all the protein were solubilized in the 75 ml of liquid, the proteinconcentration in the supernatant would be: 2.21 g/75 ml≈2.95%.

Note that the supernatants also include the digestive and exogenousenzymes. In order to determine the solubility, the protein contributionfrom the digestive and exogenous enzymes should be subtracted from theprotein concentrations in the supernatants whenever possible.% protein from the pancreatin (X mg/g diet) and pepsin (Y U/g diet)=((Xmg pancreatin/g diet×10 g diet×0.69×100%)/(1000 mg/g×75 g))+((YUpepsin/g diet×10 g diet×0.57×100%)/(539 U/mg×1000 mg/g×75 g)),

where 0.69 and 0.57 refer to the protein contents in the pancreatin andpepsin preparations used (i.e. 69% of the pancreatin, and 57% of thepepsin is protein as determined by the Kjeldahl method referred toabove).% protein from exogenous enzymes (Z mg EP/g diet)=(Z mg EP/g diet×10 gdiet×100%)/(1000 mg/g×75 g)% protein corrected in supernatant=% protein in supernatant asanalyzed−(% protein from digestive enzymes+% protein from exogenousenzymes)Protein solubilization (%)=(% protein corrected insupernatant×100%)/2.95%

The results below show that the protease derived from Nocardiopsis sp.NRRL 18262 has a significantly better effect on protein solubilizationas compared to the blank, and as compared to the Bacillus sp. NCIMB40484 protease.

Enzyme Solubilized P (% of total) SD n Blind (no exogenous enzymes)73.8^(c) 0.87 10 + the protease derived from 77.5^(a) 0.50 10Nocardiopsis sp. NRRL 18262 + the protease derived from 75.6^(b) 1.52 5Bacillus sp. NCIMB 40484 + FLAVOURZYME ™ 74.1^(c) 0.23 4 ^(a,b,c)Valueswithin a column not sharing a common superscript letter aresignificantly different, P < 0.05. SD is standard deviation; n is thenumber of observations.

EXAMPLE 5 Degradation of the Lectin SBA and the Soybean Bowman-Birk andKunitz Inhibitors

The ability of the proteases from Nocardiopsis sp. NRRL 18262 andBacillus sp. NCIMB 40484 to hydrolyze soybean agglutinin (SBA) and thesoy Bowman-Birk and Kunitz trypsin inhibitors was tested.

Pure SBA (Fluka 61763), Bowman-Birk Inhibitor (Sigma T-9777) or KunitzInhibitor (Trypsin Inhibitor from soybean, Boehringer Mannheim 109886)was incubated with the protease for 2 hours, 37° C., at pH 6.5(protease: anti-nutritional factor=1:10, based on A₂₈₀). Incubationbuffer: 50 mM dimethyl glutaric acid, 150 mM NaCl, 1 mM CaCl₂, 0.01%Triton X-100, pH 6.5.

The ability of the proteases to degrade SBA and the protease inhibitorswas estimated from the disappearance of the native SBA or trypsininhibitor bands and appearance of low molecular weight degradationproducts on SDS-PAGE gels. Gels were stained with Coomassie blue andband intensity determined by scanning.

The results, as % of anti-nutritional factor degraded, are shown inTable 4 below.

It is contemplated that the ability to degrade the anti-nutritionalfactors in soy can also be estimated by applying the Western techniquewith antibodies against SBA, Bowman-Birk Inhibitor or Kunitz Inhibitorafter incubation of soybean meal with the candidate proteases (see WO98/56260).

TABLE 4 Bowman-Birk Kunitz Protease derived from SBA Inhibitor InhibitorNocardiopsis sp. NRRL 18262 75 25 100 Bacillus sp. NCIMB 40484 21 41 100

EXAMPLE 6 Effects of Acid-Stable Nocardiopsis Proteases on the GrowthPerformance of Broiler Chickens

The trial is carried out in accordance with the official Frenchinstructions for experiments with live animals. Day-old broiler chickens(‘Ross PM3’), separated by sex, are supplied by a commercial hatchery.

The chickens are housed in wire-floored battery cages, which are kept inan environmentally controlled room. Feed and tap water is provided adlibitum.

On day 8, the chickens are divided by weight into groups of 6 birds,which are allocated to either the control treatment, receiving theexperimental diet without enzymes, or to the enzyme treatment, receivingthe experimental diet supplemented with 100 mg enzyme protein of theprotease per kg feed.

Each treatment is replicated with 12 groups, 6 groups of each sex. Thegroups are weighed on days 8 and 29. The feed consumption of theintermediate period is determined and body weight gain and feedconversion ratio are calculated.

The experimental diet is based on maize starch and soybean meal (44%crude protein) as main ingredients (Table 5). The feed is pelleted (dieconfiguration: 3×20 mm) at about 70° C. An appropriate amount of theprotease is diluted in a fixed quantity of water and sprayed onto thepelleted feed. For the control treatment, adequate amounts of water areused to handle the treatments in the same way.

For the statistical evaluation, a two factorial analysis of variance(factors: treatment and sex) is carried out, using the GLM procedure ofthe SAS package (SAS Institute Inc., 1985). Where significant treatmentseffects (p<0.05) are indicated, the differences between treatment meansare analyzed with the Duncan test. An improved weight gain, and/or animproved feed conversion, and/or improved nutritive value of soybeanmeal is expected (taking into consideration that maize starch is ahighly digestible ingredient).

REFERENCES

EEC (1986): Directive de la Commission du 9 avril 1986 fixant la méthodede calcul de la valeur énérgetique des aliments composés destinés à lavolaille. Journal Officiel des Communautés Européennes, L130, 53-54

SAS Institute Inc. (1985): SAS® User's Guide, Version 5 Edition. CaryN.C.

TABLE 5 Composition of the experimental diet Ingredients (%): Maizestarch 45.80 Soybean meal 44¹ 44.40 Tallow 3.20 Soybean oil 1.00DL-Methionine 0.18 MCP 0.76 Salt 0.05 Binder 1.00 Vitamin and mineralpremix 3.55 Avatec ® 15% CC² 0.06 Analyzed content: Crude protein (%)19.3 ME, N-corrected (MJ/kg)³ 12.2 Crude fat (%) 5.3 ¹analyzed content:90.6% dry matter, 45.3% crude protein, 2.0% crude fat, 4.9% crude fiber²corresponded to 90 mg lasalocid-Na/kg feed as anticoccidial ³calculatedon the basis of analyzed nutrients content (EC-equation; EEC, 1986)Supplier of Feed Ingredients

Maize starch: Roquettes Fréres, F-62136 Lestrem, France

Soybean meal 44: Rekasan GmbH, D-07338 Kaulsdorf, Germany

Tallow: Fondoirs Gachot SA, F-67100 Strasbourg, France

Soybean oil: Ewoco Sarl, F-68970 Guemar, France

DL-Methionine: Produit Roche SA, F-92521 Neuilly-sur-Seine, France

MCP: Brenntag Lorraine, F-54200 Toul, France

Salt: Minoterie Moderne, F-68560 Hirsingue, France

Binder: Minoterie Moderne, F-68560 Hirsingue, France

Premix (AM vol chair NS 4231): Agrobase, F-01007 Bourg-en-Bresse, France

Avatec: Produit Roche SA, F-92521 Neuilly-sur-Seine, France

EXAMPLE 7 Premix and Diets for Turkey and Salmonids Supplemented withAcid-Stable Nocardiopsis Proteases

A premix of the following composition is prepared (content per kilo):

5000000 IE Vitamin A 1000000 IE Vitamin D3 13333 mg Vitamin E 1000 mgVitamin K3 750 mg Vitamin B1 2500 mg Vitamin B2 1500 mg Vitamin B6 7666mg Vitamin B12 12333 mg Niacin 33333 mg Biotin 300 mg Folic Acid 3000 mgCa-D-Panthothenate 1666 mg Cu 16666 mg Fe 16666 mg Zn 23333 mg Mn 133 mgCo 66 mg I 66 mg Se 5.8% Calcium

To this premix the protease from Nocardiopsis sp. NRRL 18262 is added(prepared as described in Example 2), in an amount corresponding to 10 gprotease enzyme protein/kg.

Pelleted turkey starter and grower diets with a composition as shown inthe below table (on the basis of Leeson and Summers, 1997 butrecalculated without meat meal by using the AGROSOFT®, optimisationprogram) and with 100 mg protease enzyme protein per kg are prepared asfollows:

Milled maize, Soybean meal, Fish-meal and Vegetable fat are mixed in acascade mixer. Limestone, calcium phosphate and salt are added, togetherwith the above premix in an amount of 10 g/kg diet, followed by mixing.The resulting mixture is pelleted (steam conditioning followed by thepelleting step).

Starter diet, g/kg Grower, g/kg Finisher Ingredient Maize 454.4 612.5781.0 Soybean meal 391 279 61.7 Fish meal 70 29.9 70 Vegetable fat 21 2146 Limestone 19 16.9 9 Calcium phosphate 30 25.9 16.8 Salt (NaCl) 2 2 2Vitamin and mineral premix 10 10 10 Lysine 1.3 1.49 Methionine 1.3 1.33.6 Calculated nutrients Crude protein g/kg 279 213 152 Metabolizableenergy MJ/kg 12.3 12.7 14.1 Calcium, g/kg 15.8 12.7 9 AvailablePhosphorus, g/kg 8.2 6.4 4.6 Lysine, g/kg 17.6 12.8 7.5 Methionine, g/kg6.1 4.9 6.9

Two diets for Salmonids are also prepared, as generally outlined above.The actual compositions are indicated in the Table below (compiled fromRefstie et al (1998), Aquaculture, vol. 162, p. 301-302). The estimatednutrient content is recalculated by using the Agrosoft® feedoptimisation program.

The protease derived from Nocardiopsis alba, prepared as described inExample 2, is added to the diets in an amount corresponding to 100 mgprotease enzyme protein per kg.

Conventional diet Alternative diet with with fish meal soybean mealIngredient Wheat 245.3 75.2 Fish meal 505.0 310.0 Soybean meal — 339.0Fish oil 185.0 200.0 DL-Methionine 13.9 23.0 Mono-Calcium phosphate —2.0 Vitamin and Mineral premix + 50.8 50.8 pellet binder and astaxanthinCalculated nutrients (fresh weight basis) Crude protein g/kg 401 415Crude fat g/kg 232 247 Metabolizable energy MJ/kg 16.9 16.5 Calcium,g/kg 13.9 9.8 Phosphorus, g/kg 10.8 9.0 Lysine, g/kg 27.7 26.7Methionine, g/kg 24.4 31.6

EXAMPLE 8 Determination of Purity of Protease-Containing Enzyme Products

The purity of protease-containing enzyme products, e.g. proteasepreparations such as commercial multi-component enzyme products, can bedetermined by a method based on the fractionation of theprotease-containing enzyme product on a size-exclusion column.Size-exclusion chromatography, also known as gel filtrationchromatography, is based on a porous gel matrix (packed in a column)with a distribution of pore sizes comparable in size to the proteinmolecules to be separated. Relatively small protein molecules candiffuse into the gel from the surrounding solution, whereas largermolecules will be prevented by their size from diffusing into the gel tothe same degree. As a result, protein molecules are separated accordingto their size with larger molecules eluting from the column beforesmaller ones.

Protein Concentration Assay.

The protein concentration in protease-containing enzyme products isdetermined with a BCA protein assay kit from PIERCE (identical to PIERCEcat. No. 23225). The sodium salt of Bicinchoninic acid (BCA) is astable, water-soluble compound capable of forming an intense purplecomplex with cuprous ions (Cu¹⁺) in an alkaline environment. The BCAreagent forms the basis of the BCA protein assay kit capable ofmonitoring cuprous ions produced in the reaction of protein withalkaline Cu²⁺ (Biuret reaction). The color produced from this reactionis stable and increases in a proportional fashion with increasingprotein concentrations (Smith, P. K., et al. (1985), AnalyticalBiochemistry, vol. 150, pp. 76-85). The BCA working solution is made bymixing 50 parts of reagent A with 1 part reagent B (Reagent A is PIERCEcat. No. 23223, contains BCA and tartrate in an alkaline carbonatebuffer; reagent B is PIERCE cat. No. 23224, contains 4% CuSO₄*5H₂O). 300microliter sample is mixed with 3.0 ml BCA working solution. After 30minutes at 37° C., the sample is cooled to room temperature and A₄₉₀ isread as a measure of the protein concentration in the sample. Dilutionsof Bovine serum albumin (PIERCE cat. No. 23209) are included in theassay as a standard.

Sample Pre-Treatment.

If the protease-containing enzyme product is a solid, the product isfirst dissolved/suspended in 20 volumes of 100 mM H₃BO₃, 10 mM3,3′-dimethylglutaric acid, 2 mM CaCl₂, pH 6 (Buffer A) for at least 15minutes at 5° C., and if the enzyme at this stage is a suspension, thesuspension is filtered through a 0.45 micron filter to give a clearsolution. The solution is from this point treated as a liquidprotease-containing enzyme product.

If the protease-containing enzyme product is a liquid, the product isfirst dialyzed in a 6-8000 Da cut-off SpectraPor dialysis tube (cat.no.132 670 from Spectrum Medical Industries) against 100 volumes of BufferA+150 mM NaCl (Buffer B) for at least 5 hours at 5° C., to removeformulation chemicals that could give liquid protease-containing enzymeproducts a high viscosity, which is detrimental to the size-exclusionchromatography.

The dialyzed protease-containing enzyme product is filtered through a0.45 micron filter if a precipitate was formed during the dialysis. Theprotein concentration in the dialyzed enzyme product is determined withthe above described protein concentration assay and the enzyme productis diluted with Buffer B, to give a sample ready for size-exclusionchromatography with a protein concentration of 5 mg/ml. If the enzymeproduct has a lower than 5 mg/ml protein concentration after dialysis,it is used as is.

Size-Exclusion Chromatography.

A 300 ml HiLoad26/60 Superdex75pg column (Amersham Pharmacia Biotech) isequilibrated in Buffer B (Flow: 1 ml/min). 1.0 ml of theprotease-containing enzyme sample is applied to the column and thecolumn is eluted with Buffer B (Flow: 1 ml/min). 2.0 ml fractions arecollected from the outlet of the column, until all of the applied samplehas eluted from the column. The collected fractions are analyzed forprotein content (see above Protein concentration assay) and for proteaseactivity by appropriate assays. An example of an appropriate assay isthe Suc-AAPF-pNA assay (see Example 2B). Other appropriate assays aree.g. the CPU assay (se Example 1), and the Protazyme AK assay (seeExample 2D). The conditions, e.g. pH, for the protease activity assaysare adjusted to measure as many proteases in the fractionated sample aspossible. The conditions of the assays referred to above are examples ofsuitable conditions. Other suitable conditions are mentioned above inthe section dealing with measurement of protease activity. A proteinpeak with activity in one or more of the protease assays is defined as aprotease peak. The purity of a protease peak is calculated as theprotein amount in the peak divided with the total protein amount in allidentified protease peaks.

The purity of a protease-containing enzyme product is calculated as theamount of protein in the acid-stable protease peak divided with theprotein amount in all identified protease peaks using the aboveprocedure.

1. A method for hydrolyzing anti-nutritional factors in a vegetable,comprising adding an acid-stable protease to an anti-nutritional factor,wherein the protease comprises an amino acid sequence having an identityof at least 90% to SEQ ID NO:
 1. 2. The method of claim 1, wherein theanti-nutritional factor is derived from soybean.
 3. The method of claim1, wherein the anti-nutritional factor is lectin soybean agglutinin. 4.The method of claim 1, wherein the anti-nutritional factor is a trypsininhibitor.
 5. The method of claim 1, wherein the protease has an aminoacid sequence having an identity of at least 95% to SEQ ID NO:
 1. 6. Amethod of treating vegetable proteins, comprising adding (i) anacid-stable protease and (ii) a phytase to vegetable proteins, whereinthe protease comprises an amino acid sequence having an identity of atleast 90% to SEQ ID NO:
 1. 7. The method of claim 6, wherein thevegetable protein is derived from legumes.
 8. The method of claim 6,wherein the vegetable protein is derived from cereals.
 9. The method ofclaim 6, wherein the vegetable protein is derived from soybean.
 10. Themethod of claim 6, wherein the protease has an amino acid sequencehaving an identity of at least 95% to SEQ ID NO:
 1. 11. A method forincreasing solubilization of vegetable proteins, comprising adding anacid-stable protease to vegetable proteins, wherein the proteasecomprises an amino acid sequence having an identity of at least 90% toSEQ ID NO:
 1. 12. The method of claim 11, wherein the vegetable proteinis derived from legumes.
 13. The method of claim 11, wherein thevegetable protein is derived from cereals.
 14. The method of claim 11,wherein the vegetable protein is derived from soybean.
 15. The method ofclaim 11, wherein the protease has an amino acid sequence having anidentity of at least 95% to SEQ ID NO: 1.